As a method to conduct intracellular protein synthesis ex vivo, such as in a test tube, an in vitro cell-free protein synthesis method has been widely researched, wherein ribosome, for example, and other components necessary for protein synthesis are extracted from an organism (herein sometimes referred to as “wheat embryo extract for cell-free protein synthesis”) and used (Patent Documents 1, 2, 3, 4 and 5).
The cell-free protein synthesis system is a useful method, which keeps performance comparable to the system in a living cell concerning accuracy and speed of translation reaction, and can provide a target protein without carrying out a complicated purification step. Therefore, the more effective application of the synthesis system for industrial uses needs, in addition to increasing synthesis efficiencies, providing a wheat embryo extract-containing solution for use in synthesis and a ready-made type wheat embryo extract-containing solution steadily and at a retained high quality.
Meanwhile, the infection caused by a pathogen including viruses, even though it has been overcome to some extent by progress in chemotherapy and so on, is still a big threat to human, as can be seen from year-by-year epidemic of influenza, the occurrence of novel infectious diseases such as AIDS, E. Coli O157, SARS, West Nile virus, and Ebola hemorrhagic fever or from the reoccurrence of tuberculosis which was believed to have been overcome for a while. Under these circumstances, it has recently been demonstrated that a protease inhibitor to HIV virus is effective as an anti-AIDS drug, and it is put into practical use. The research for an infectious disease treatment medicament to target a protease which is indispensable for viral proliferation is believed to be more important in future.
At present, in a commonly conducted research on a protease inhibitor, an E. Coli or the like is treated by genetic recombination technology to produce a protease protein, which is then coexisted with the substrate protein and a test substance to search for an effective substance as an inhibitor using a substrate cleaving activity as an indicator. However, the production of a protein from a pathogen by genetic recombination needs a large scale experiment facility at a level of P3 and P4 is required and subjects itself to many regulatory constraints. In addition, a researcher can not perfectly be exempted from the risk to be infected with those pathogens even if protected by sufficient facilities.
Further, according to a conventional method, a drug, which is determined by an in vitro experiment to have a protease inhibitory activity, can in some cases not inhibit viral proliferation. It is suggested that the drug according to the conventional method is purified to complete its folding so that it may have a different structure to affect substrate-specificity.
In addition, recent infectious diseases of concern, SARS (severe acute respiratory syndrome) is an emerging infectious disease appeared in early 2003, and has affected 8,447 people and deprived 811 lives worldwide as of the end of June in 2003. SARS is characterized by high fever, malaise, ague, headache and dyspnoea, and progressed to cause interstitial pneumonia, requiring intubation and mechanical respiration. Currently, the fatality rate in the SARS infected is as high as approximately 15%, and it is believed that the infection route is mainly by direct contact although other routes cannot be fully excluded. From the many evidences, as a novel coronavirus exists in persons infected with SARS, the responsible pathogen for SARS is believed to be a novel coronavirus (SARS-CoV). Further, an essential proteinase for SARS-CoV's self-reproduction is found out, and it is pointed out that the catabolic enzyme shows important functions in the viral life cycle while causing SARS's symptoms (Nonpatent Document 2). Therefore, a proteinase for SARS-CoV (SARS 3CLpro) is considered to be a drug target effective in SARS treatment, and diverse researches have been conducted.
Until now, the following pharmacological researches on SARS have been proceeded by a number of groups. Study on candidate inhibitors of SARS 3CLpro in molecular modeling for the three dimensional structural analysis of SARS 3CLpro's crystalline structure, which is a primary proteinase for SARS-CoV (Nonpatent Document 1). Study on candidate drugs to SARS in molecular modeling for the binding mechanism between SARS 3CLpro and ligands (Nonpatent Document 2). They have not experimentally examined SARS 3CLpro and inhibitors thereto, and not reached to practical application.
Further, as the study of an inhibitor to the pathogen using a cell-free protein synthesis system, a method for screening an inhibitor to inhibit autodigestion is proposed wherein a rhinovirus protease is expressed in a cell-free protein synthesis system derived from rabbit's reticulocytes. However, that screening method is not practical because, for example, to screen enormous candidate drugs using a cell-free protein synthesis system derived from rabbit's reticulocytes needs a plenty of synthesis solution to obtain, has a problem in cost, and further requires a tracer experiment to execute using a radioisotope for the screening detection due to the slight amount of expressed protein in the synthesis system of interest (Nonpatent Document 3).
In spite of the above situation in SARS study, currently no effective drug for treating SARS has been available. The reasons are as follows: SARS-CoV has been recently identified to be the pathogen of SARS. Furthermore, to study such a highly-fatal virus as SARS pathogen, the experiment is difficult to manage because of constrains from biohazard. Moreover, it has been believed that a bioactive protein expressed from a decoded genomic sequence is difficult to screen while it keeps activity, because of problems from its conformation and posttranslational modification.    [Patent Document 1] Japanese Patent Application Laid-open No. Hei 6-98790    [Patent Document 2] Japanese Patent Application Laid-open Hei 6-225783    [Patent Document 3] Japanese Patent Application Laid-open Hei 7-194    [Patent Document 4] Japanese Patent Application Laid-open Hei 9-291    [Patent Document 5] Japanese Patent Application Laid-open Hei 7-147992    [Patent Document 6] International Patent Application PCT/US98/25742    [Nonpatent Document 1] Anand K, Ziebuhr J, Wadhwani P, Mesters J R, Hilgenfeld R.    Coronavirus main proteinase (3Clpro) structure:basis for design of anti-SARS drugs. Science. 2003 Jun. 13; 300(5626):1763-7. Epub 2003 May 13.    [Nonpatent Document 2] Kuo-Chen Chou, Dong-Qing Wei, and Wei-Zhu Zhong.    Binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against SARS. Biochemical and Biophysical Research Communications 308 (2003) 148-151    [Nonpatent Document 3] BEVERLY A. HEINZ, JOSEPH TANG, JEAN M. LABUS, FREDERICK W. CHADWELL, STEPHEN W. KARLDOR, AND MARLYS HAMMOND    Simple In Vitro Translation Assay To Analyze Inhibitors of Rhinovirus Proteases    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, January 1996, p. 267-270